GSM7925727: FGC0743_s_1_TTAGGC; Escherichia coli; OTHER
N-terminally tagged GST-FUS was overexpressed in BL21(DE3)RIL E. coli. The E. coli cells were then lysed by sonication on ice in PBS and protease inhibitors (cOmplete, EDTA-free, Roche Applied Science). The protein was purified over Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) and eluted from the beads using FUS assembly buffer (50mM Tris-HCl pH 8, 200mM trehalose, 1mM DTT, and 20mM reduced glutathione). RNA that was bound to GST-FUS during protein purification was extracted by adding DNase I and then Proteinase K to the sample followed by phenol-chloroform extraction, and precipitation in 100% ethanol with 70% ethanol wash. For preparing cDNA libraries for high-throughput sequencing, we used the NEBNext® Small RNA Library Prep Set for Illumina® and followed the manufacturer's instructions. Library quality was checked with the Agilent 2100 BioAnalyzer. The sample was sequenced on the Illumina HiSeq2000 platform using hiSeq-50SR.